In the B Cell Focus our overall goals are to 1) design Env immunogens that will elicit both difficult-to-induce broad neutralizing antibodies, and also 2) induce easier-to-induce protective antibodies. The goal is to induce both types of antibodies in most vaccinated subjects. Specific aims include: Aim 1. To define the nature of protective systemic and mucosal immunity in vaccinated subjects. Aim 2. To design novel gp120/gp140 immunogens that induces protective mucosal and systemic antibody responses to HlV-1. Aim 3. To define host factors that may limit the induction of broadly neutralizing antibodies. Aim 4. To design immunogens that target unmutated ancestor and intermediate antibodies of the maturation pathways of protective anti-HlV-1 Env antibodies. Aim 5. To use structural biological information and technology to design immunogens. Thus, the B Cell Focus will use recombinant monoclonal antibody technology to study unique mucosal samples from those vaccinated with current vaccines; will work to define the types of antibodies and their protective nature induced at mucosal sites (Aim 1); will test new, more antigenic and immunogenic transmitted/founder Env immunogens in Aim 2, will determine tolerance and other immunoregulatory host factors that control induction of difficult-to-induce broadly neutralizing antibodies (BnAbs) (Aim 3); will isolate clonal lineages of BnAbs to define precursor antibodies to use as templates upon which to design new immunogens for driving unusual or complex maturation pathways (Aim 4), and will solve the near atomic resolution structure of the membrane associated trimer by cryoEM for rationale vaccine design (Aim 5). The work of the B Cell Focus builds on discoveries made over the past 6 years in CHAVI, and aims to overcome the current roadblocks preventing inducing protective antibodies in the majority of vaccinated subjects.